Many of the problems currently being studied in molecular biology and biochemistry share a common factor: they are governed by essential molecular interactions, which are often protein-protein interactions. Important examples are the identification and functional characterization of novel gene products, the dissection of proteins into structural or functional motifs and the testing of hypotheses about the physical basis of protein-protein interactions, whether in naturally-occurring proteins or in designed products. The highly successful yeast two-hybrid assay has been demonstrated to be very effective in genome-wide screening for interacting proteins (1,2). However, the yeast two-hybrid system has limitations when applied to the mapping of protein-protein interactions in higher eukaryotes. It would be an advantage either to screen or, at a minimum, to perform followup studies of interacting partners directly in the context of the cell in which the proteins function and in the correct subcellular compartment. For example, in the case of mammalian proteins, a mammalian cell instead of a yeast cell would be the preferred context for screening and biologically validating protein-protein interactions.
In addition, it would be an advantage to be able to construct screens based on a variety of library types and sources, including natural diverse libraries such as cDNA libraries or single-chain antibody libraries; synthetic diverse libraries such as peptide libraries; or defined libraries such as full-length gene collections. These needs could potentially be met by a protein-protein interaction technology that could be performed in vivo, in any cellular context, and with the ability to engineer the assay properties and assay stringency.
Also, as the study of interacting partners is a “two-dimensional” problem influenced by variations in either partner, it would be advantageous in certain cases to pan a library of proteins not against a single bait protein, but against a second library of proteins. To date, no large-scale library-vs-library selection of protein-protein interactions has been reported, because the available strategies are not amenable to this in any practical way.
Finally, it would be an advantage to have a screening technology that is suitable for scale-up and automation with a choice of instrumentation platforms.
PCA involves tagging proteins with polypeptide fragments derived by rationally dissecting a reporter. If two proteins that are tagged with complementary fragments interact, the fragments are brought into close proximity. The complementary fragments can then fold into an active conformation and re-constitute the activity of the reporter from which the fragments were derived. At its basic level, PCA is a general and flexible strategy that allows detection of protein-protein interactions in vivo and also allows measurement of the association and dissociation of protein-protein complexes in real time. PCA has unique features that make it a useful tool for molecular and cell biology:                Molecular interactions are detected directly, not through secondary events such as transcription activation.        A variety of detection methods can be used, including cell growth (e.g. survival-selection), fluorescent, colorimetric, luminescent and phosphorescent detection, depending on the choice of reporter.        Proteins can be expressed in the relevant cellular context, reflecting the native state of the protein with the correct post-translational modifications other cellular proteins that are necessary, directly or indirectly, for controlling the interactions that are being measured by the PCA.        Because protein-protein complexes can be quantitated with PCA in the live cell context, immediate functional validation of protein-protein interactions can be achieved following library screening.        
The present invention describes the uses of PCA in screening for protein-protein interactions in vivo and in validating the protein-protein interactions identified in the screens. Strategies, examples, and suitable reporters are provided for a range of cell types including bacterial, mammalian and human cells. Examples are provided for peptide libraries, cDNA libraries, and defined (full-length) gene libraries. The ability to choose among many different reporters allows a choice of readouts for the detection of a protein-protein interaction, including survival-selection, fluorescence, luminescence or phosphorescence or color. Examples are provided for survival-selection and fluorescence assays. The methods can be applied to library-vs.-library screening, bait-vs.-library screening, and interaction mapping of a full-length gene library. Moreover, these methods are amenable to scale-up and automation with a choice of instrumentation platforms for low-cost, large-scale screening.